Stem Cell Reports
Article
The MicroRNA-371 Family as Plasma Biomarkers for Monitoring
Undifferentiated and Potentially Malignant Human Pluripotent Stem Cells
in Teratoma Assays
Daniela C.F. Salvatori,1,5,*Lambert C.J. Dorssers,2,5Ad J.M. Gillis,2Gemma Perretta,3Ton van Agthoven,2 Maria Gomes Fernandes,1Hans Stoop,2Jan-Bas Prins,1J. Wolter Oosterhuis,2Christine Mummery,4 and Leendert H.J. Looijenga2,*
1Central Laboratory Animal Facility, Leiden University Medical Center, Einthovenweg 20, Leiden 2333 ZC, the Netherlands
2Department of Pathology, Laboratory for Experimental Patho-Oncology, Erasmus MC Cancer Institute, Be-432A, PO Box 2040, 3000 CA Rotterdam, the
Netherlands
3Fondazione Guido Bernardini, Via Manfredo Camperio, 10, 20123 Milano, Italy
4Department of Anatomy & Embryology, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, the Netherlands 5Co-first author
*Correspondence:d.c.f.salvatori@lumc.nl(D.C.F.S.),l.looijenga@erasmusmc.nl(L.H.J.L.)
https://doi.org/10.1016/j.stemcr.2018.11.002
SUMMARY
Predicting developmental potency and risk of posttransplantation tumor formation by human pluripotent stem cells (hPSCs) and their derivatives largely rely on classical histological analysis of teratomas. Here, we investigated whether an assay based on microRNAs (miRNA) in blood plasma is able to detect potentially malignant elements. Several hPSCs and human malignant germ cell tumor (hGCT) lines were investigated in vitro and in vivo after mouse xenografting. The multiple conventional hPSC lines generated mature teratomas, while xenografts from induced hPSCs (hiPSCs) with reactivated reprogramming transgenes and hGCT lines contained undifferentiated and potentially malignant components. The presence of these elements was reflected in the mRNA and miRNA profiles of the xenografts with OCT3/4 mRNA and the miR-371 and miR-302 families readily detectable. miR-371 family members were also iden-tified in mouse plasma faithfully reporting undifferentiated elements in the xenografts. This study demonstrated that undifferentiated and potentially malignant cells could be detected in vivo.
INTRODUCTION
The teratoma assay is still the most widely accepted method for defining pluripotency of human stem cell lines. Putative human pluripotent stem cells (hPSCs) are injected into extra-uterine sites of immune-deficient mice where they form xenografts. These tumors are often highly het-erogeneous, composed of derivatives of all three embryonic germ layers, ectoderm, endoderm, and mesoderm. They are then referred to as teratomas, while teratocarcinomas contain also malignant embryonal carcinoma (EC) cells
(Buta et al., 2013; Damjanov and Andrews, 2016). Yolk
sac (YS), an extraembryonic endodermal tissue, has also been described in teratomas and teratocarcinomas. The teratoma assay is laborious, animal-based, essentially qual-itative, and rarely standardized, with inconsistencies re-ported in methodology and histopathological evaluation (Muller et al., 2010). To determine the clinical safety of hPSC-based therapies, the ability to detect undifferentiated PSCs and potentially malignant cells is extremely impor-tant (Allison et al., 2018; Damjanov and Andrews, 2016). In vitro tests such as PluriTest or ScoreCard, that describe the expression of pluripotency or lineage markers have been proposed as alternatives to the teratoma assay, but these in vitro assays may miss detecting the malignant
potential hPSCs (Allison et al., 2018; Bouma et al., 2017; Muller et al., 2011; Tsankov et al., 2015). Another test, called TeratoScore, based on quantifying gene expression in xenografts, has shown good agreement with the tera-toma assay with respect to identification of tissues derived from the three germ layers, but ultimately only the tera-toma assay has been shown to detect both pluripotency and malignancy (Allison et al., 2018; Avior et al., 2015).
In human pathology, teratomas belong to a class of com-plex neoplasms called germ cell tumors (human germ cell tumor [hGCT]). During histopathological evaluation of hGCTs, the presence of undifferentiated cells and YS ele-ments are a clear indication of malignancy. The biological significance of these elements is still debated in the context of mouse xenografts derived from injection of hPSCs. In the WHO pathology classification of tumors, various hGCT types are distinguished, of which postpubertal (type II) malignant hGCTs are of particular interest. Histo-logically, these are referred to as either seminoma (SE) or non-seminoma (NS). NS can represent all cell lineages present during early embryogenesis, including somatic as well as extra-somatic tissues such as YS tumor and chorio-carcinoma. They all originate from EC cells, the malignant counterpart of embryonic stem cells (ESCs) (Andrews et al., 1980, 1984; Pera et al., 1989). EC cells, such as ESCs, are
characterized by expression of OCT3/4 and SOX2 (de Jong
et al., 2008; Looijenga et al., 2003), two transcription
factors also used to generate induced pluripotent stem cells (iPSCs) (Takahashi and Yamanaka, 2006). This demon-strates the similarities between hGCTs and hiPSCs (
Cun-ningham et al., 2012). All malignant hGCTs, with the
exception of teratomas (TE), are characterized by high expression of a defined set of miRNAs (Gillis et al., 2007; Looijenga et al., 2007). These include miR-371a-3p, -372-3p, -373--372-3p, and -367--372-3p, which are also highly expressed in hESCs and hiPSCs (Barroso-del Jesus et al., 2009; Lip-china et al., 2012; Parr et al., 2016; Zhang et al., 2013). These miRNAs have been shown to be specific and sensitive (liquid) biomarkers for malignant hGCTs in serum, plasma, as well as cerebrospinal fluid of patients (Belge et al., 2012; Dieckmann et al., 2012; Gillis et al., 2013; Lea˜o et al., 2018; Murray et al., 2011; Syring et al., 2015; van Agthoven et al., 2017).
In this study, we investigated whether plasma miRNAs could serve as facile biomarkers to detect undifferentiated and potentially malignant cell types in xenografts and in plasma samples derived from mice injected with hPSC
lines and hGCT cell lines. A set of hPSC lines and hGCT cell lines was investigated after in vitro culture and in vivo after being xenografted into mice. Each cell line and corresponding xenografts were examined for mRNA and miRNA expression. Patient-derived hGCTs were included for comparison. In addition, mouse plasma miRNA profiles were monitored over time during xenograft formation. The results demonstrated that the histological detection of undifferentiated and malignant components in the xenografts could be predicted with high accuracy on the basis of plasma miR-371a-3p levels. We hypothesize that this could be of value in detecting later malignancy should it arise in patients who have undergone transplantation of differentiated hPSCs as therapy.
RESULTS
Design of the Study
The study design is shown schematically inFigure 1. Mul-tiple hESC and hiPSC lines and (malignant) hGCT cell lines were investigated (Table S1B). Each was examined for mRNA and miRNA expression after culture in vitro and in vivo after xenografting to mice, to investigate the extent of similarity. The xenografts were also examined (immuno) histologically by experienced pathologists. Matched serial plasma samples were profiled for miRNA expression during xenograft development and at the endpoint of the experi-ment when mice were euthanized. A list of all abbrevia-tions used is given inTable S1A.
Histological Characterization of the Xenografts
Xenografts were morphologically characterized in H&E-stained sections and by immunohistochemical detection of OCT3/4, NANOG, CD30, SOX2, SOX17, and GPC3: OCT3/4 and NANOG (Figure 2) to detect undifferentiated hPSCs and potentially malignant elements (Ulbright et al., 2014). CD30 is a survival factor and marker of trans-formed human EC cells (Herszfeld et al., 2006). SOX17 is informative for SE cells, SOX2 for EC, both being co-ex-pressed with OCT3/4 and NANOG (Cheng et al., 2007; Eini et al., 2014; Hart et al., 2005; Looijenga, 2009; Looi-jenga et al., 2003; Rijlaarsdam et al., 2011). Glypican-3 (GPC3) identifies YS elements, associated with loss of SOX2 and gain of SOX17 (in the absence of OCT3/4 and NANOG) (Looijenga, 2009; Looijenga et al., 2003; Ulbright et al., 2014). Representative H&E-stained sections are shown in Figure 2. Xenografts derived from H9 hESCs and the H9Hyb hESC line consisted only of fully differen-tiated teratomas, confirming the immunohistochemical findings. The Lu07 hiPSC line also consisted predomi-nantly of differentiated derivatives of the three germ layers
Figure 1. Schematic Representation of the Work Flow Performed
but had some YS elements (see below). Xenografts of all of these cell lines contained somatic tissue structures repre-senting all three germ layers (neural rosettes and retinal pigmented epithelium [ectoderm], intestinal epithelium [endoderm], cartilage, bone, fat, and muscle [mesoderm]) and they were negative for the pluripotency markers OCT3/4 and NANOG. This is in line with previous immu-nofluorescent staining forbIII-tubulin(ectoderm), human a-fetoprotein (AFP, endoderm) and human PECAM-1 (CD31, mesoderm) (Bouma et al., 2017). By contrast, the 2102Ep xenografts consisted of EC, i.e., they had not un-dergone somatic differentiation, as expected from their tumor origin. Xenografts from the Lu07 + Dox hiPSC line, in which the reprogramming genes had been reacti-vated by giving the mice doxycycline (Dox) in their drink-ing water, were mostly composed of EC components resembling the 2102EpL xenografts, corresponding with their immunohistochemical profiles (positive for OCT3/4,
SOX2, and CD30), with small scattered areas positive for GPC3 (i.e., YS), as also found in the Lu07 hiPSC line without Dox. The TCam-2-derived xenograft showed mixed pattern of SE, EC, and YS elements, again corre-sponding to the immunohistochemical profile showing heterogeneous staining for OCT3/4/SOX2/CD30 (for EC), GPC3 (for YS), and OCT3/4 and SOX17 (SE). In summary, H9 and H9Hyb hESC lines gave rise to xenografts that were composed entirely of differentiated derivatives of the three germ layers; they were therefore classified as TE (Figure 2). The Lu07 hiPSC xenografts were predominantly composed of tissues derived from the three germ layers representing TE, but they also showed small scattered groups of GPC3-positive cells, i.e., YS differentiation. These YS cells were also found in the Lu07 + Dox xenografts with reactivated reprogramming factors, although these were otherwise predominantly composed of undifferentiated cells identified as EC cells.
mRNA Profiling
Previously, we reported the mRNA profiles of xenografts from the Lu07 + Dox hiPSC line with reactivated reprog-ramming transgenes and 2102EpL which completely lacked differentiated elements in histology (Bouma
et al., 2017). To extend these datasets and to highlight
the similarities between these and hGCTs, we included an additional series of hGCT-derived cell lines and pri-mary testicular hGCTs, representing the various histolog-ical elements and types of hGCTs, i.e., EC, YS tumors and SEs (Table S2). To capture the major similarities between these 74 samples, principal-component analysis (PCA) of the top differentiating genes (4% of total) was performed. The 3D PCA plot clearly resolved the cell cultures, xeno-grafts, and hGCT groups (Figure 3). The Lu07 + Dox hiPSC xenografts were separated from the xenografts derived from H9 and H9Hyb hESCs and Lu07 hiPSCs without Dox. Clustering using the top 99 differential probes confirmed the overall PCA patterns (Figure S1) and also showed the extensive overlap in expression between hGCTs and the Lu07 + Dox hiPSC xenografts. Multiple genes associated with ‘‘stem cell properties’’ and ‘‘malig-nancy’’ were expressed in the Lu07 + Dox hiPSC xeno-grafts. These mRNA profiles indicated that the majority
Figure 2. Histopathological Characterization of Xenografts Derived from Different hPSC and hGCT Cell Lines
(A–G) Representative pictures of H&E and immunohistochemistry of xenografts derived from hPSCs (A) H9, (B) H9 + Dox, (C) H9Hyb, (D) Lu07, (E) Lu07 + Dox and hGCT-derived cell lines, (F) 2102EpL, and (G) TCam-2. H&E-stained xenografts (A–D) show differentiated tissue areas representing derivatives of mesoderm (Meso), ectoderm (Ecto), and endoderm (Endo). Scale bars, 200mm. Representative images of immunohistochemistry for the pluripotency markers OCT4 and specific markers for hGCTs SOX17, SOX2, CD30, and GPC3 are shown. Nuclei were counterstained with hematoxylin only. Scale bars, 100mm (for the immunohistochemistry images) (seeTable S3for antibody list). (H) Summary of presence of tissues derived from the three embryonic germ layers, undifferentiated embryonal carcinoma (EC) cells, yolk sac (YS) elements, and seminoma (SE) components in the xenograft. Notice that Lu07, although negative for all other markers, showed small groups of cells positive for GPC3 (i.e., YS differentiation).
Figure 3. Principal-Component Analysis of mRNA Expression of hPSCs, hPSC-Derived Xenografts, hGCT-Derived Cell Lines and Xenografts and Primary Testicular hGCTs
of the stem cell markers were not present in xenografts in which there was full differentiation into mature TE, while they remained high in Lu07 + Dox hiPSC xenografts, re-flecting retention of their undifferentiated histology. The Lu07 + Dox hiPSC tumors therefore resembled hGCTs and the 2102EpL xenografts which also continued to ex-press many pluripotency markers. Constitutive exex-pression of pluripotency genes thus confers classic features of hGCTs in these hiPSC lines.
miRNA Profiling
The miRNA expression profile of the xenografts was determined using TaqMan low-density arrays (TLDA cards A&B). In total, 768 individual miRNAs were as-sayed in the xenografts and compared with the profiles of a set of control mouse organs, human EC cell lines (NCCIT, 2102Ep, NT2), the SE cell line TCam-2, as well as the undifferentiated hPSCs (Lu07 + Dox, Lu07, H9, H9+Dox, and H9Hyb) cultured in vitro. The resulting heatmap (Figure 4) showed the relative levels of the top differentiating miRNAs (n = 57) in these samples and indicated higher levels of the different members of the miR-371, miR-302, and miR-518 families in the un-differentiated Lu07 hiPSCs, with or without Dox, Lu07 + Dox_T-derived xenografts, and the NCCIT, 2102Ep, NT2, and TCam-2 cells and xenografts. Xeno-grafts consisting only of mature TE completely lacked expression of these miRNAs. Many of these miRNA fam-ilies are located in the primate-specific chromosome 19 miRNA cluster (C19MC,Figure 4), which has been impli-cated in ESC function and specifically associated with malignant hGCTs (Barroso-del Jesus et al., 2009; Lip-china et al., 2012; Parr et al., 2016; Zhang et al., 2013) but not with TE (Flor et al., 2016; Gillis et al., 2007;
Looijenga et al., 2007; Shen et al., 2018). A number of
downregulated miRNAs was also identified, including members of the miR-34 cluster.
In light of these patterns of miRNAs and their apparent reflection of the histological constitution of cell lines in vitro and in vivo, mouse plasma miRNA levels were examined at the endpoint of the xenograft, using the TLDA card assays, and compared with t = 0 (before injec-tion) as control. The same miRNA families (miR-371, miR-302, and miR-518) distinguished plasma samples
of mice injected with the Lu07, H9, H9 + Dox (dox con-trol), and H9Hyb cell lines from mice injected with the (malignant) Lu07 + Dox, 2102EpL, and TCam-2 cell lines
(Figure 5), being significantly expressed in the latter
group. In light of the high levels of significance of these distinguishing markers, we then examined plasma samples collected during xenograft development to determine when the onset of potential malignant growth was detectable. For each mouse investigated, 40 Ct values of miRNA levels in all plasma samples and the calculated tumor volume (mm3) were evaluated (Figures 6andS2).
In mice transplanted with Lu07 (ST70) or Lu07 + Dox (ST72) cells, plasma levels of the control miRNA (miR-20a and 30b, both cross-reactive with mouse miRNAs) remained stable throughout the experiment, while the miR-371/2/3 and -367 accumulated in the plasma during time. In the case of Lu07 + Dox mice, the time of detec-tion was earlier and the levels were much higher than for the condition without Dox. In both cases, detection of circulating miRNAs in plasma preceded the detection of the actual tumor by physical palpation. Additional time series using mice xenografted with Lu07 hiPSCs, 2102EpL, and TCam-2 cells, confirmed these findings
(Figure S2). In all cases, the selected target miRNAs
were detected before the tumor became physically man-ifest and the levels compared with control plasma were generally >10 Ct (i.e., over 1,000-fold difference). The finding of late plasma accumulation of miR-371/2/3 and -367 in mice transplanted with Lu07 cells without Dox in the drinking water could be associated with the histological finding of a minor YS element in the corre-sponding xenograft (Figure 2). To resolve this further, all available additional endpoint plasma samples of mice with xenografts of H9 and H9Hyb hESCs and Lu07 hiPSCs were evaluated for miR-371/373 and the control miR-20a and -30b (Figure 7). The results showed that mice with Lu07 hiPSCs without the addition of Dox have low plasma levels of miR-371 and -373, while addi-tion of Dox strongly enhanced these levels only in the Lu07-transplanted mice and not in any without the Dox-inducible reprogramming element in the genome. The H9 and H9hybrid hESC lines also showed very low plasma levels of these miRNAs at the endpoint of the xenograft.
Figure 4. miRNA Expression Profile Comparison between hPSCs and hGCT-Derived Cell Lines and Respective Xenografts
DISCUSSION
In this study we determined mRNA and miRNA profiles of cell lines, xenografts, and circulating miRNAs in matched plasma of mice injected with multiple hPSCs and hGCTs with various histological composition, and related these to the expected teratoma and teratocarcinoma characteris-tics. We demonstrated that expression levels of the miR-371, -302, and C19MC families revealed the presence of undifferentiated and malignant features in the Lu07 + Dox hiPSCs, 2102EpL and TCam-2, both in cell cultures and derivative tumors. Histologically, 2102EpL and Lu07 + Dox hiPSC tumors were similar to each other, being mainly composed of EC-like cells expressing the pluripo-tency markers OCT3/4 and SOX2. TCam-2 xenografts were diagnosed as mixed tumors containing SE, EC, and YS elements. The designation ‘‘mixed tumor’’ for the TCam-2 xenografts is in line with previously published data showing that subcutaneous TCam-2 xenotransplanta-tion initiates reprogramming into an EC-like fate
(Nettersheim et al., 2016). The H9 and H9Hyb, hESCs,
and Lu07 hiPSCs, formed TE containing differentiated cells of the three germ layers and consistently lacked undifferen-tiated cells (shown by histology and confirmed by immu-nohistochemistry by the absence of OCT3/4 and NANOG
expression); the Lu07 hiPSC line in the absence of Dox somewhat unexpectedly showed YS areas positive for GPC3. In this set of xenografts and terminal plasma sam-ples, levels of the miR-371, -302, and C19MC families were below detection. These findings overlapped with pre-viously reported human clinical pathology in which miR-371-3, -302, and C19MC family were found expressed in all testicular hGCTs except TE (Flor et al., 2016; Gillis et al., 2007; Looijenga et al., 2007; Shen et al., 2018). In particular, the miR-371 family has already proven to be a reliable serum biomarker for testicular hGCTs in patients (Belge et al., 2012; Dieckmann et al., 2012; Gillis et al., 2013; Lea˜o et al., 2018; Murray et al., 2011; Syring et al.,
2015; van Agthoven et al., 2017), and we showed here
that it can be used to monitor tumor development with minimum invasiveness without the need to terminate the experiment. hGCTs much like as those derived here from the injection of hPSCs, can present highly heteroge-neous histology. Although TE are assumed to be non-malig-nant, it is recognized that they may contain a variable number of residual undifferentiated cells, creating risk of malignancy (Damjanov and Andrews, 2016; Terenziani
et al., 2015). However, malignancy features are rarely
discussed when interpreting results from the teratoma assay. Teratomas are widely described in the stem cell field
Figure 5. MicroRNA Expression Profile of the Xenograft Endpoint Plasma
as benign tumors composed of mature somatic tissue in contrast to teratocarcinomas, the malignant counterparts that include EC (Damjanov and Andrews, 2007; Muller et al., 2010). This terminology contrasts with that used by clinical pathologists because the description and analysis of the malignant component is too limited; indeed EC is not the only possible malignant component of hPSC-derived tumors (Oosterhuis and Looijenga, 2005) and this should be taken into account in considering potential safety issues with transplantation of hPSC derivatives in regenerative medicine. The initiating hPSCs have the intrinsic capacity to differentiate into all three germ layers of the embryo proper, but also into extraembryonic ele-ments such as YS. Presence of YS is mostly neglected during analysis of teratomas. It is noteworthy that this extraem-bryonic endodermal tissue is associated with malignant progression in pediatric TE (Gonzalez-Crussi et al., 1978;
Heiftz, 1998; O’Connor and Norris, 1994), and it shows
similarities with the behavior of ESCs (Oosterhuis and
Looijenga, 2005), including expression of the miR-371
and -302 families (Murray et al., 2011).
Following cell transplantation, plasma samples collected from mice injected with Lu07, Lu07 + Dox, 2102EpL, and TCam-2, showed increasing levels of miR-371/2/3 and -367 targets with time. The observation that the levels plateaued in most xenografted mice prior to the maximum measured tumor size, suggests that production of miRNAs is balanced by clearing from the circulation as was also observed in patients after surgical removal of clinically manifest hGCTs (Dieckmann et al., 2017; van Agthoven
et al., 2017). However, an alternative explanation might
be that larger tumors may be less effective in releasing miRNAs in the circulation, possibly due to reduced growth and/or viability. This is, however, unlikely based on the correlation found between the miRNA levels and the over-all tumor load (Dieckmann et al., 2012; van Agthoven and
Looijenga, 2017). Interestingly, production of these
miRNAs was, at the end of the experiment, detected in mice transplanted with Lu07 hiPSCs without Dox. Although this was initially unexpected based on the avail-able results derived from tissue and plasma miRNA profiling (Figures 4 and 5), it is likely explained by the presence of a minor YS component in the corresponding xenografts. We showed in earlier work that a fraction of Lu07 and Lu07 + Dox hiPSCs contained an additional short arm of chromosome 12 (inFigure S1) (Bouma et al., 2017). The correlation between karyotypic abnormalities and their influence to malignant potential is still under discus-sion (Allison et al., 2018).
In the Lu07 + Dox, 2102EpL, and TCam-2 malignant xenografts, downregulation of the miR-34 cluster was identified (Figure 4). Members of miR-34 family have been shown to be p53 induced, and they act as tumor sup-pressors regulating key cancer pathways (Rokavec et al., 2014). This observation is therefore in line with the initial model in which the miR-371 family was found to be an alternative mechanism for inactivation of the P53 pathway in hGCTs (Voorhoeve et al., 2006).
In conclusion, this study demonstrated the similarities between hESCs, hiPSCs, and (malignant) hGCTs regarding their histological composition, mRNA, miRNA, and pro-tein expression profiles. This combined approach resulted
Figure 6. Time Course of miR-371, -372, -373, and -367 Accumulation in the Mouse Plasma Samples of Two Representative Mice Injected with Lu07 (Right Panel) and Lu07 + Dox Cells (Left Panel)
in an informative pre-clinical model, allowing investiga-tion of various aspects of pluripotency regulainvestiga-tion and tumor risk prediction. Hence, it shows that evaluation of circulating miRNAs in the plasma represents a real-time refinement of the histological analysis of the teratoma assay detecting undifferentiated, and possibly malignant elements. Specifically, the elevated levels in liquid biopsies of a defined set of embryonic miR, being miR-371, -302, and C19MC family members, is of ultimate interest because of their link to the presence of undifferentiated and potentially malignant cell populations. The patterns found in the mouse xenograft model mimic those in hGCT patients. This observation may be expected to have clinical value in development of a minimally invasive method for monitoring development of malignancy in
the context of hESC and hiPSC applications in regenerative medicine.
EXPERIMENTAL PROCEDURES
Cell Lines and Patient Samples
Cell lines used in this study are summarized inTable S1B. hPSC lines: hESC lines H9 and H9Hyb (H9 clone Hybrid_6), and hiPSC LUMC007iCTRL01 (Lu07) (Bouma et al., 2017) were kindly supplied by C. Freund, LUMC. The hiPSC Lu07 was generated from skin fibroblasts using a Dox-inducible lentivirus encoding mouse cDNAs for OCT3/4, SOX2, KLF4, and MYC separated by three different 2A peptides (TetO-FUW-OSKM) and a lenti-virus carrying the tetracycline-controllable transactivator (FUW-M2rtTA, plasmids from Addgene) as already reported (Bouma et al., 2017). Lu07 was cultured in the absence (Lu07) or presence (Lu07 + Dox) of 1 mg/mL Dox (Sigma). As a control, H9 cells were also cultured in the absence (H9) or presence of Dox (H9 + Dox).
Five aneuploid (malignant) hGCT cell lines were used: TCam-2 representing SE (Mizuno et al., 1993), and four cell lines represent-ing EC (NT2 [NTera-2];Andrews et al., 1984), NCCIT (Teshima et al., 1988), and two batches of 2102Ep (Andrews et al., 1980; Josephson et al., 2007; Wang et al., 1980) [kindly supplied by P. Andrews, Sheffield, UK]). 2102Ep was maintained at the Erasmus Medical Center and the 2102EpL cultured at the LUMC, and used for the xenograft experiments.
Cell lines were characterized for genomic constitution (karyotyp-ing) as described previously (Bouma et al., 2017) or verified for identity by STR genotyping against the DSMZ (https://www. dsmz.de/) database (hGCT cell lines).
In addition, for the human patient samples, a total of 25 primary testicular hGCTs were included in the study. This series included: 15 pure SE and 10 NS of which the histological composition (EC, TE, YS tumor, or SE) is indicated in the sample name (Table S2). Histological diagnosis was performed by expert pathol-ogists. Use of patient tissue samples remaining after diagnosis was approved for research by the Medical Ethical Committee of the EMC (the Netherlands), permit no. 02.981. This included permis-sion to use the secondary tissue without further consent. Samples were used according to the ‘‘Code for Proper Secondary Use of Human Tissue in The Netherlands’’ developed by the Dutch Federation of Medical Scientific Societies (FMWV, version, 2002; update 2011).
Teratoma Assay
Cells (13 106) were injected subcutaneously in the flank region of 8- to 10-week-old male NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ, Charles River). Tumor growth was monitored weekly by palpation, and mice were euthanized when tumors reached a volume of%2 cm3. Tumor size was measured using a digital caliper. Tumor volume was calculated according to the method reported before (Feldman et al., 2009). Animal experiments were approved by the Dutch Central Commis-sion for Animal experimentation (Centrale Commissie voor Dierproeven).
Figure 7. Levels of miR-371 and miR-373 for Endpoint Plasma Samples of Mice Injected with H9, H9Hyb, Lu07, and Lu07 + Dox Cell Lines
When indicated, Dox (2 mg/mL, Sigma-Aldrich) was provided in the drinking water of the mice together with sucrose (10 mg/mL, Sigma-Aldrich) 1 week prior to injections and was renewed every 2 days for the duration of the experiment. Drink-ing water without Dox contained sucrose only. Blood samples were obtained from the animals before injections and every 7–14 days via tail bleeding. In some instances due to animal wel-fare considerations the bleeding interval was minimally 7 days but did not go beyond 14 days; for each bleeding, the maximum amount of blood did not exceed 7.5% of the circulating blood volume. Blood was collected in a 1.5-mL Eppendorf tube (containing 7.5mL of heparin 1,000 m/mL), centrifuged at 14,000 rpm for 5 min at 4C after which plasma was stored at80C until analysis.
Morphological and Immunohistochemical Examination of Xenografts
Macroscopically representative parts of the tumors were snap frozen (liquid nitrogen) as well as fixed in formalin and embedded in paraffin (FFPE). H&E and various immunohistochemical stain-ings (antibodies specified in Table S3) were performed (frozen and FFPE) for evaluation as described previously (Bouma et al., 2017; van Casteren et al., 2009). Evaluation was performed independently by an experienced human pathologist in hGCTs (J.W.O.) and a scientist (L.H.J.L.), as well as one European board-certified veterinary pathologist (D.C.F.S.). Histological classification was based on the most recent WHO classification ( Ul-bright et al., 2016).
mRNA and miRNA Analysis
RNA from xenografts (_T) was isolated, labeled, and hybridized onto the Illumina Human HT-12 v4 array (Illumina, San Diego, CA, USA) and processed as described previously (Bouma et al., 2017). In addition, total mRNA from cell lines (_C), and the hGCT was profiled using Illumina HumanHT-12 v4, as described previously (Van Der Zwan et al., 2013). Sample description is provided inTable S2. Array data are available from ArrayExpress (accession E-MTAB-6312). For miRNA profiling, total RNA was extracted from cells and xenografts using the TRIzol reagent (In-vitrogen) and miRVana miRNA Isolation Kit (Thermo Fischer Scientific), respectively. All reagents for miRNA isolation and quantification were obtained from Thermo Fisher Scientific (www.thermofisher.com/). Total RNA was also prepared from a mix of organs (brain, heart, kidney, testis, lungs, striated muscles, lymph nodes, small, and large intestine) of an age-, sex-, and strain-matching mouse following the same procedure adopted for the xenografts. Plasma (p) of xenografted or control mice (10–50mL) was processed using the TaqMan miRNA ABC purifica-tion Bead Kit human panel A or B (PN: 4473087/4473088, each capturing 384 miRNAs) using the supplied protocol (PN: 4473439) and as described previously for human serum (Gillis et al., 2013). miRNAs were converted into cDNA using the spe-cific megaplex primers (PN: 4399966/4399967) and the reverse transcription kit (PN; 3466596), pre-amplified (plasma) and quan-titated on TaqMan low-density arrays (two 384-well Microfluids TLDA cards, PN: 4398965/4398966) on a TaqMan 7900 thermo-cycler using the supplier protocols (PN: 4399721). TaqMan
miRNA array output data (sds files) were uploaded in the Thermo Fisher Cloud App (https://www.thermofisher.com/ mysso/loginDisplay) and analyzed using defined threshold set-tings for each individual miRNA. Cq values were exported (Table S4), and globally normalized using QbasePlus (Biogazelle, Zwij-naarde, Belgium). Individual assays for specific miRNAs (hsa-miR-371a-3p, hsa-miR-372-3p, hsa-miR-373-3p, hsa-miR-367-3p, hsa-miR-20a-5p, and hsa-miR-30b-5p; Thermo Fisher Scientific) were run on a TaqMan 7500 fast thermocycler as described previ-ously (van Agthoven et al., 2017). A non-human miRNA spike-in ath-miR-159a was added to the plasma samples for quality control of miRNA isolation and calibration (van Agthoven and Looijenga, 2017).
Analysis of mRNA and miRNA Data
Heatmaps of mRNA and miRNA data were generated in R using the ‘‘pheatmap’’ clustering software package using default settings. For mRNA clustering, ‘‘correlation’’ was used as the distance measure for columns. PCA was performed using prcomp from the R Stats package.
ACCESSION NUMBERS
Deposited mRNA profiling data can be found in the ArrayExpress database (www.ebi.ac.uk/arrayexpress), under accession number ArrayExpress: E-MTAB-6312.
SUPPLEMENTAL INFORMATION
Supplemental Information includes two figures and four tables and can be found with this article online athttps://doi.org/10. 1016/j.stemcr.2018.11.002.
AUTHOR CONTRIBUTIONS
D.C.F.S., L.C.J.D., and L.H.J.L. designed and analyzed the experi-ments and wrote the manuscript in collaboration with the other authors. A.J.M.G., G.P., T.v.A., M.G.F., H.S., and J.B.P. carried out the experiments. J.W.O., D.C.F.S., and L.H.J.L. reviewed the pathol-ogy. C.M. and L.H.J.L. supervised and provided the facilities. All au-thors read and approved the final manuscript.
ACKNOWLEDGMENTS
This work is part of the research program ‘‘Meer Kennis met Minder Dieren’’ (More Knowledge with Less Animals) with project number 2017104947IZONMW, financed by the Netherlands Organisation for Scientific Research (NWO). Ton van Agthoven is supported by the Dutch Cancer Society (grant number 13-6001). Special thanks to the LUMC hiPSC core facility staff for support. We also thank Saskia Maas and Sophie Geerhardt for daily care of the mice and the histopathology work. We thank Prof Bart Janssen (GenomeScan, the Netherlands) for in kind support of the micro-array assays.
REFERENCES
Allison, T.F., Andrews, P.W., Avior, Y., Barbaric, I., Benvenisty, N., Bock, C., Brehm, J., Bru¨stle, O., Damjanov, I., Elefanty, A., et al. (2018). Assessment of established techniques to determine devel-opmental and malignant potential of human pluripotent stem cells. Nat. Commun. 9, 1925.
Andrews, P., Damjanov, I., Simon, D., Banting, G., Carlin, C., Dracopoli, N., and Føgh, J. (1984). Pluripotent embryonal carci-noma clones derived from the human teratocarcicarci-noma cell line Tera-2. Differentiation in vivo and in vitro. Lab. Invest. 50, 147–162.
Andrews, P.W., Bronson, D.L., Benham, F., Strickland, S., and Knowles, B.B. (1980). A comparative study of eight cell lines derived from human testicular teratocarcinoma. Int. J. Cancer 26, 269–280.
Avior, Y., Biancotti, J.C., and Benvenisty, N. (2015). TeratoScore: as-sessing the differentiation potential of human pluripotent stem cells by quantitative expression analysis of teratomas. Stem Cell Reports 4, 967–974.
Barroso-del Jesus, A., Lucena-Aguilar, G., and Menendez, P. (2009). The miR-302-367 cluster as a potential stemness regulator in ESCs. Cell Cycle 8, 394–398.
Belge, G., Dieckmann, K.P., Spiekermann, M., Balks, T., and Buller-diek, J. (2012). Serum levels of microRNAs miR-371-3: a novel class of serum biomarkers for testicular germ cell tumors? Eur. Urol. 61, 1068–1069.
Bouma, M.J., van Iterson, M., Janssen, B., Mummery, C.L., Salva-tori, D.C.F., and Freund, C. (2017). Differentiation-defective human induced pluripotent stem cells reveal strengths and limita-tions of the teratoma assay and in vitro pluripotency assays. Stem Cell Reports 8, 1340–1353.
Buta, C., David, R., Dressel, R., Emgard, M., Fuchs, C., Gross, U., Healy, L., Hescheler, J., Kolar, R., Martin, U., et al. (2013). Reconsi-dering pluripotency tests: do we still need teratoma assays? Stem Cell Res. 11, 552–562.
Cheng, L., Sung, M.T., Cossu-Rocca, P., Jones, T., MacLennan, G., Jong, J.D., Lopez-Beltran, A., Montironi, R., and Looijenga, L. (2007). OCT4: biological functions and clinical applications as a marker of germ cell neoplasia. J. Pathol. 211, 1–9.
Cunningham, J.J., Ulbright, T.M., Pera, M.F., and Looijenga, L.H.J. (2012). Lessons from human teratomas to guide development of safe stem cell therapies. Nat. Biotechnol. 30, 849.
Damjanov, I., and Andrews, P.W. (2007). The terminology of teratocarcinomas and teratomas. Nat. Biotechnol. 25, 1212, discus-sion 1212.
Damjanov, I., and Andrews, P.W. (2016). Teratomas produced from human pluripotent stem cells xenografted into immuno-deficient mice - a histopathology atlas. Int. J. Dev. Biol. 60, 337–419.
de Jong, J., Stoop, H., Gillis, A.J., Hersmus, R., van Gurp, R.J., van de Geijn, G.J., van Drunen, E., Beverloo, H.B., Schneider, D.T., Sher-lock, J.K., et al. (2008). Further characterization of the first seminoma cell line TCam-2. Genes Chromosomes Cancer 47, 185–196.
Dieckmann, K.P., Radtke, A., Spiekermann, M., Balks, T., Matthies, C., Becker, P., Ruf, C., Oing, C., Oechsle, K., Bokemeyer, C., et al. (2017). Serum levels of microRNA miR-371a-3p: a sensitive and specific new biomarker for germ cell tumours. Eur. Urol. 71, 213–220.
Dieckmann, K.P., Spiekermann, M., Balks, T., Flor, I., Loning, T., Bullerdiek, J., and Belge, G. (2012). MicroRNAs miR-371-3 in serum as diagnostic tools in the management of testicular germ cell tu-mours. Br. J. Cancer 107, 1754–1760.
Eini, R., Stoop, H., Gillis, A.J., Biermann, K., Dorssers, L.C., and Looijenga, L.H. (2014). Role of SOX2 in the etiology of embryonal carcinoma, based on analysis of the NCCIT and NT2 cell lines. PLoS One 9, e83585.
Feldman, J.P., Goldwawwer, R., Mark, S., Schwartz, J., and Orion, I. (2009). A mathematical model for tumor volume eval-uation using two-dimensions. J. Appl. Quantitative Methods 4, 455–462.
Flor, I., Spiekermann, M., Loning, T., Dieckmann, K.P., Belge, G., and Bullerdiek, J. (2016). Expression of microRNAs of C19MC in different histological types of testicular germ cell tumour. Cancer Genomics Proteomics 13, 281–289.
Gillis, A.J., Rijlaarsdam, M.A., Eini, R., Dorssers, L.C., Bier-mann, K., Murray, M.J., Nicholson, J.C., Coleman, N., Die-ckmann, K.P., Belge, G., et al. (2013). Targeted serum miRNA (TSmiR) test for diagnosis and follow-up of (testicular) germ cell cancer patients: a proof of principle. Mol. Oncol. 7, 1083–1092.
Gillis, A.J., Stoop, H.J., Hersmus, R., Oosterhuis, J.W., Sun, Y., Chen, C., Guenther, S., Sherlock, J., Veltman, I., Baeten, J., et al. (2007). High-throughput microRNAome analysis in human germ cell tumours. J. Pathol. 213, 319–328.
Gonzalez-Crussi, F., Winkler, R.F., and Mirkin, D.L. (1978). Sacro-coccygeal teratomas in infants and children: relationship of histology and prognosis in 40 cases. Arch. Pathol. Lab. Med. 102, 420–425.
Hart, A.H., Hartley, L., Parker, K., Ibrahim, M., Looijenga, L.H.J., Pauchnik, M., Chung, W.C., and Robb, L. (2005). The pluripotency homeobox gene NANOG is expressed in human germ cell tumors. Cancer 104, 2092–2098.
Heiftz, S.A.e.a. (1998). Immature teratomas in children: pathologic considerations: a report from the Combined Pediatric Onco-logy Group/Children’s Cancer Group. Am. J. Surg. Pathol. 22, 1115–1124.
Herszfeld, D., Wolvetang, E., Langton-Bunker, E., Chung, T.L., Fil-ipczyk, A.A., Houssami, S., Jamshidi, P., Koh, K., Laslett, A.L., Mi-chalska, A., et al. (2006). CD30 is a survival factor and a biomarker for transformed human pluripotent stem cells. Nat. Biotechnol. 24, 351–357.
Josephson, R., Ording, C.J., Liu, Y., Shin, S., Lakshmipathy, U., Toumadje, A., Love, B., Chesnut, J.D., Andrews, P.W., Rao, M.S., et al. (2007). Qualification of embryonal carcinoma 2102Ep as a reference for human embryonic stem cell research. Stem Cells 25, 437–446.
Hansen, A., et al. (2018). Serum miRNA predicts viable disease after chemotherapy in patients with testicular nonseminoma germ cell tumor. J. Urol. 200, 126–135.
Lipchina, I., Studer, L., and Betel, D. (2012). The expanding role of miR-302–367 in pluripotency and reprogramming. Cell Cycle 11, 1517–1523.
Looijenga, L. (2009). Human testicular (non)seminomatous germ cell tumours: the clinical implications of recent pathobiological insights. J. Pathol. 218, 146–162.
Looijenga, L.H., Stoop, H., de Leeuw, H.P., de Gouveia Brazao, C.A., Gillis, A.J., van Roozendaal, K.E., van Zoelen, E.J., Weber, R.F., Wolffenbuttel, K.P., van Dekken, H., et al. (2003). POU5F1 (OCT3/4) identifies cells with pluripotent potential in human germ cell tumors. Cancer Res. 63, 2244–2250.
Looijenga, L.H.J., Gillis, A.J.M., Stoop, H., Hersmus, R., and Oosterhuis, J.W. (2007). Relevance of microRNAs in normal and malignant development, including human testicular germ cell tumours. Int. J. Androl. 30, 304–315.
Mizuno, Y., Gotoh, A., and Kamidono, S. (1993). Establishment and characterization of a new human testicular germ cell tumor cell line (TCam-2). Nihon Hinyokika Gakkai Zasshi 84, 1211– 1218.
Muller, F.J., Goldmann, J., Loser, P., and Loring, J.F. (2010). A call to standardize teratoma assays used to define human pluripotent cell lines. Cell Stem Cell 6, 412–414.
Muller, F.J., Schuldt, B.M., Williams, R., Mason, D., Altun, G., Papa-petrou, E.P., Danner, S., Goldmann, J.E., Herbst, A., Schmidt, N.O., et al. (2011). A bioinformatic assay for pluripotency in human cells. Nat. Methods 8, 315–317.
Murray, M.J., Halsall, D.J., Hook, C.E., Williams, D.M., Nicholson, J.C., and Coleman, N. (2011). Identification of microRNAs from the miR-371373 and miR-302 clusters as potential serum biomarkers of malignant germ cell tumors. Am. J. Clin. Pathol. 135, 119–125.
Nettersheim, D., Heimsoeth, A., Jostes, S., Schneider, S., Feller-meyer, M., Hofmann, A., and Schorle, H. (2016). SOX2 is essen-tial for in vivo reprogramming of seminoma-like TCam-2 cells to an embryonal carcinoma-like fate. Oncotarget 7, 47095– 47110.
O’Connor, D.M., and Norris, H.J. (1994). The influence of grade on the outcome of stage I ovarian immature (malignant) teratomas and the reproducibility of grading. Int. J. Gynecol. Pathol. 13, 283–289.
Oosterhuis, J.W., and Looijenga, L.H.J. (2005). Testicular germ-cell tumours in a broader perspective. Nat. Rev. Cancer 5, 210.
Parr, C.J.C., Katayama, S., Miki, K., Kuang, Y., Yoshida, Y., Mori-zane, A., Takahashi, J., Yamanaka, S., and Saito, H. (2016). MicroRNA-302 switch to identify and eliminate undifferentiated human pluripotent stem cells. Sci. Rep. 6, 32532.
Pera, M.F., Cooper, S., Mills, J., and Parrington, J.M. (1989). Isolation and characterization of a multipotent clone of human embryonal carcinoma cells. Differentiation 42, 10–23.
Rijlaarsdam, M.A., van Herk, H.A.D.M., Gillis, A.J.M., Stoop, H., Jenster, G., Martens, J., van Leenders, G.J.L.H., Dinjens, W.,
Hoogland, A.M., Timmermans, M., et al. (2011). Specific detection of OCT3/4 isoform A/B/B1 expression in solid (germ cell) tumours and cell lines: confirmation of OCT3/4 specificity for germ cell tumours. Br. J. Cancer 105, 854.
Rokavec, M., Li, H., Jiang, L., and Hermeking, H. (2014). The p53/miR-34 axis in development and disease. J. Mol. Cell Biol. 6, 214–230.
Shen, H., Shih, J., Hollern, D.P., Wang, L., Bowlby, R., Tickoo, S.K., Thorsson, V., Mungall, A.J., Newton, Y., Hegde, A.M., et al. (2018). Integrated molecular characterization of testicular germ cell tumors. Cell Rep. 23, 3392–3406.
Syring, I., Bartels, J., Holdenrieder, S., Kristiansen, G., Muller, S.C., and Ellinger, J. (2015). Circulating serum miRNA (miR-367-3p, miR-371a-3p, miR-372-3p and miR-373-3p) as bio-markers in patients with testicular germ cell cancer. J. Urol. 193, 331–337.
Takahashi, K., and Yamanaka, S. (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–676.
Terenziani, M., D’Angelo, P., Inserra, A., Boldrini, R., Bisogno, G., Babbo, G.L., Conte, M., Dall’ Igna, P., De Pasquale, M.D., Indolfi, P., et al. (2015). Mature and immature teratoma: a report from the second Italian pediatric study. Pediatr. Blood Cancer 62, 1202–1208.
Teshima, S., Shimosato, Y., Hirohashi, S., Tome, Y., Hayashi, I., Kanazawa, H., and Kakizoe, T. (1988). Four new human germ cell lines. Lab. Invest. 59, 328–336.
Tsankov, A.M., Akopian, V., Pop, R., Chetty, S., Gifford, C.A., Da-heron, L., Tsankova, N.M., and Meissner, A. (2015). A qPCR ScoreCard quantifies the differentiation potential of human pluripotent stem cells. Nat. Biotechnol. 33, 1182–1192.
Ulbright, M.B., Balzer, A.B., Berney, D.M., Epstein, J.I., and Guo, C. (2016). Germ cell tumours. In WHO Classification of Tumours of the Urinary System and Male Genital Organs, H. Moch, P.A. Humphrey, M.T. Ulbright, and V.E. Reuter, eds. (IARC), pp. 189–226.
Ulbright, T.M., Tickoo, S.K., Berney, D.M., and Srigley, J.R. (2014). Best practices recommendations in the application of immunohis-tochemistry in testicular tumors: report from the International Society of Urological Pathology consensus conference. Am. J. Surg. Pathol. 38, e50–e59.
van Agthoven, T., Eijkenboom, W.M.H., and Looijenga, L.H.J. (2017). microRNA-371a-3p as informative biomarker for the follow-up of testicular germ cell cancer patients. Cell Oncol. (Dordrecht) 40, 379–388.
van Agthoven, T., and Looijenga, L.H.J. (2017). Accurate primary germ cell cancer diagnosis using serum based microRNA detection (ampTSmiR test). Oncotarget 8, 58037–58049.
Van Der Zwan, Y.G., Stoop, H., Rossello, F., White, S.J., and Looi-jenga, L.H. (2013). Role of epigenetics in the etiology of germ cell cancer. Int. J. Dev. Biol. 57, 299–308.
Voorhoeve, P.M., le Sage, C., Schrier, M., Gillis, A.J., Stoop, H., Nagel, R., Liu, Y.P., van Duijse, J., Drost, J., Griekspoor, A., et al. (2006). A genetic screen implicates miRNA-372 and miRNA-373 as oncogenes in testicular germ cell tumors. Cell 124, 1169–1181.
Wang, N., Trend, B., Bronson, D.L., and Fraley, E.E. (1980). Nonrandom abnormalities in chromosome 1 in human testicular cancers. Cancer Res. 40, 796–802.
